edited by GEORGE L. GILBERT Denison University Granville. Ohio 43023
Magic Sand: Modelling the Hydrophobic Effect and Reversed-Phase Liquid Chromatography
CHECKED BY
Richard Cornellus
Lebanon Vallsv Colleae Annville, PA lf003
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A product cal1ed"MagicSand" was marketed by Wham-0 Manufacturing Company for a period of time and is still available in some toy stores1. The effects that you observe when Magic Sand is added t o water are dramatic, but the Mneic is not where the maeic resides: It resides in the -..--- Sand underappreciated and amazing (if not magic) properties of water. It is the "universal nonsolvent" ~ r o ~ e r t i of e swater that give rise to the effects demonstrated here, effects that mav escapeus if we think of water as the "universalsolvent". FO; example, the hydrophobic effect that arises when very insoluble covalent species interact with water is probably responsible for the formation of biological membranes and the evolution of life as we know it. I n a sense, "Human beings were invented by water as a device for transporting itself from one place to another" as Tom Robbins says ( I ) . In chemistry courses we emphasize the importance of the ability of water to dissolve nutrients and metabolites and carry them throueh ~ - ~ ~~e~~ - cell membranes. But without the hvdro~hobic effect, there would probably be no cell membranes! only in water. as a result of the hvdro~hobiceffect. are the DNA doubie helix and native proteins stable. In addition to the solvent properties of water, teachers should emphasize the importance of, and consequences of, insolubility in water. T h e ~roceduresdescribed here are meant to reveal the important "nonsolvent" properties of water through its in: teraction with Magic Sand and other synthetic silica derivatives, especially those with bonded organic moities. These materials have been developed along with modern liquid chromatographic techniquesthat utilize them as unusual (if not magic) stationary phases. I n a sense, all of these synthetic silicas could be called "maeic sands". Here are sueeestions for revealing the magic of water in combination with some interesting silica derivatives. ~~~~~~
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stroys the hydrophobic effect. In reversed-phase HPLC, it can be largely a hydrophobic bond between the eluite (analyte) and the very nonpolar stationary phase that allows separation (see below). The eluite may not really bond to the stationary phase, hut rather be "pushed out" of the aqueous mobile phase and forced to agglomerate on the column.. . iust as Maeic Sand aeelomerates (with itself). .... Methanol isa very strong eluant in reversed phase HPLC hecause it increaser the lipophilicity of water dramatically end decreases the hydrophobic expulsion of eluite onto the column. The next demonstration is a model of a reversed-phase liquid chromatographysystem. 5. To model a reversed-phase high-performance Liquid ehromatography (RPLC) column, coat 8 cm of one end of a 25-cm-long glass rod or tube with silicone grease, and roll the rod in yellow Magic Sand so that the sand adheres to the silicone grease. Use larger tubing or a graduated cylinder for larger scale demonstrations. The Magic Sand models the stationary phase that would normally be contained in a column. Add red3 Magic Sand to 100 mL water in a 250-mL tall-form beaker so that the magic sand floats on the surface. The red sand represents the nonpolar eluite in an aqueous eluant. Thrust the rod coated with Magic Sand through this surface layer and into the water. The red Magic Sand adheres to the yellow sand on the column! The red Magic Sand falls off when the pipet is removed from the water, or when methanol is added to the water. The "affinity" of the stationary phase for nonpolar (hydrophobic) substances in RPLC is thus a result of the rejection of hydrophohie substances by water, with other interactions contributing to a greater or lesser extent. 6. Add Magic Sand to water containing a surfactant, such as Triton X-100. Decreased surface tension allows the sand to "fall through" the surface more easily, creating smaller shapes. Stirring destroys the nonwettability of the Magic Sand. The high surface tension of water is apparently necessary to create the effects observed when Magic Sand and water are mixed. Only very effective surfactants in fairly high concentrations will destroy the Magic Sand effect. 7. Repeat the above experiments with octadecylsilylated ('LC18") silicas made for solid-phase extraction columns4or from spent reversed-phase HPLC columns. These materials float, but exhibit bulk nonwettability when forced to suhmerge with a spatula. You may also want to explore the resistance to wetting of the 5-20-pm silica gel commonly used for normal-phase liquid chromatography. Basic solutions destroy the nonwettability of the sand at about the same rate that they destroy reversed phase HPLC stationary phases, but water and common organic solvents do not affect Magic
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Demonstrations 1. Pour 100 mL of Magic Sand2into about 400 mL of water in a 600-mL beaker. Much Larger quantities may be used for large lecture halls. Note the "rabbit-intestine-like" structures that form. This is an illustrative model of the "hydrophobic effect". How important was water to the morphogenesis of biological structures? 2. Pour the mixture of water and Magic Sand slowly into an empty 600-mL beaker. The structures assumed by Magic Sand are clearly not due to attractions between its particles, just as the "hydrophobic bond" has no existence in the absence of water orsimilar liquids. In the absence of water, there is no apparent attraction! It must be the properties of water that make Magic Sand magic. How can this hypothesis be tested? 3. Pour 1M) mL of Magic Sand into about 400 mL of hexane in a 600-mL beaker. There is no magic effect. The hydrophobic bond does not exist in a "lipophilic" solvent. 4. Pour Magic Sand into methanollwater solutions of various concentrations to model the effect of an organic modifier (in this case methanol) in the primarily aqueous eluant used in reversedphase HPLC. A very small concentration of organic modifier de-
512
Journal of Chemlcal Education
Part of this wwk was presentedat the 21st Mlddle AtlantlcRegional Meeting of the American Chemical Society, Stockton State College. Pomona, NJ. May 19, 1987. Astro Sand, a product identical to Magic Sand, may be obtained from the Clifford W. Estes Co.. Inc.. P.O. Box 907. Lvndhurst. NJ
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20 lb or so (although nodefinite minimum is stated)ior S6.90tlb plus about $3 shipping. if the material is temporarily om of stock from this supplier, the author will supply % Ib (adequate Magic Sand for many demonstrations) for $5 including shipping. A substlute can be made by spraying dry sand or silica gel with ScotchGard and allowing the product to dry overnight. The checker sprayed ScotchGard on 250 mL of sand until the mass of the contents of the ScotchGard can had decreased by 110 g, and let the product dry overnight to obtain suitable material. If two colors of Magic Sand are not available, the dye from colored material can be removed with bleaches or solvents, producing a "natural" color. None of these demonstrations actually requires two colors. 'Fisher Sclentiflc supplies "Prep-Sep" extraction columns containing varlous stationary phases. J. T. Baker Chemlcal Company. Phlilipsburg. NJ 08865, and Supelco, Inc., Supelco Park, Bellefonte, PA 16823. supply similar products.
Sand except to remove the dyes that may he present. Thua s few ounces o f M w i c S a n d can he reused for many demonstration8 if i t is allowed to d r y on a paper tom1 a n d returned to the original contain-
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Dlscusslon The Hydrophobic Effect
The -~ "hvdroohobic effect" refers t o the interactions between insolubie covalent species in water, and the manner in which these interactions eive rise to suoermolecular structures or to unique conformations of a single macromolecule ~
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Fiaure 1. Schematic reoresentation showlna that discrete molecule^ o l a hydrophonk s~bsmnceIn water are s~noundadby highly adered solvsm cages When they a g g r w t e to t o m d mers. enwwy deneases as a rssu t of the release 01 water molecules Into more random aqueoLs environment This process is endothermic dna, there is a net decrease in hydrogen bonding, a,the driving forcemust be entroplc.
with water-insoluble moities. The interaction between water insoluble species in an aqueous environment is called the "hydrophobic bond". The hydrophobic bond is not an attraction (such as a van der Wads force) that would exist between the species if water were absent (2).The stability of the hydrophobic bond is aresult of the entropy decrease that would result in the surrounding water if the interaction were eliminated (that is. if thev were comoletelv solvated as mon- -~disperses ~ e c i e s ) . ~ u r ~ r ~ idisshuti& n ~ l y , of hydrocarbons in water is exothermic (3), and would thus be spontaneous (with a negative free energy, since AG = AH - TAS) if it were not for the negative entroov of dissolution. The neeative entropy changeapparently ;&ults from the formatioiof a highly ordered "cage" of water molecules around the hvdroihobic suhstance; This cage may be similar to the on& that have been identified in clathrates (4). Thecagearound a hydrophobic molecule is uniquely labile, however, a3 indicated by the large increase in heat capacity that accompanies cage formation (5).Figure 1 shows that association of two molecules will reduce the size of the ordered "cage", and give rise to a positive entropy change, so insolubility is entropically favored. Thus hvdroohobic substances aeelomerate to eive structures t h a t minimize the number of-irdered w a t e ~ m o l e c ~ s surroundine them. Breakaee of the hvdroohobic bond t o give mono&sperse hydropiohic rno1ec;les &udly leads t o the formation of more (or stronger) hvdroeen bonds in the solvent. Thus breakage of the hy&ophobic'bond is associated with a negative enthalpy change, and L e Chatelier'sprin-
Flgure 2 Scann ng e echon m crograuhs ISEM SI of Mag c Sano panic es %surface appears son' st 200X magn.1 c a l m ,a1 but shiny when tna Jana nar osen mated wlth basesolAon (01 S ~ r i a c emlcrospneres oecomo v so0 eat 30 OOOX magnillcat on lcl, bnareremoved oy treatment wlth baseas shown in ,261 E ecnon mlcro~rapnys technically aIn8cAt oecadse ot the ms.latlng strenmn of tne materla
Volume 67
Number 6
June 1990
513
cinle nredicta that hvdronhobic bonds should be less stable atloder temperatur& ~ i tobacco e mosaic virus (TMV), for examole. can be denatured bv decreasine its temnerature in wateiso'that hydrophobic bonds necessary in maintaining its normal conformation are destroved. TMV is not oreserved in the refrigerator! Some authors suggest caution in discussing the hvdrophobic effect because it is based on statisticai mech&icai arguments (6) with several assumptions and because there is a paucity of experimental work on some critical issues.
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Maaic Sand Is Not Entlrelv . Hvdroohobic . . Magic Sand does not exhibit its macroscopic (bulk) nonwettabilitv entirelv as a result of the same forces that create the true'~hydrophbbiceffect",although hulk nonwettability is a property of hydrophobic substances. .Magic Sand is apmade by depositing microspheresof silica on-a silica (sand) grain of comparatively large size. The silica microspheres probably have a small concentration of bonded alkylsilane (7). Scanning electron micrographs of Magic Sand are shown in Fieure 2. Fieures 2a and 2b show Maeic Sand particles magnified a b o u h 0 0 ~but , the particle i n i b has been treated with aqueous base t o remove the microspheres on the surface. The soft appearance of the sand in Figure 2a contrasts with the "shiny" surface revealed in Figure 2b. Figures 2c and 2d show particles at 30,000X magnification. wain untreated, and treated with base to remove surface microspheres, respectively. Water probably cannot bond to Magic Sand, even though many hydrophilic Si-OH groups exist on thesurface of the largesand particle, because of a gross surface tension effect similar to the effect that keeps droplets of water from penetrating the hair on a caterpillar's back. T o fill the cavities between silica microparticles on the Maeic Sand surface would lead to an imnossiblv large increase in surface energy, as would enlarging a droplet to fill the space between hairs on a caterpillar. This model is suggested by the diagram in Figure 3, where it is shown that a surface "bridge" of hundreds of water molecules across two silica microspgeres would require an energy equal to the product of the increase in surface area (A, cm2) and surface tension (y, dynelcm) to expand into thecavity. The behavior of Magic Sand in a vacuum appears a t first to support this model. When water is added under vacuum to Magic Sand, the nonwettability of the Sand is observed. When pressure above the water is increased to near one atmosphere, the
Sand Particle
Figure 3. Schematic representation (not to scale) of the interface between water and a Megic Sand particle. It is proposedthat the free energy (dG= 7dA) necessary to expand the water surtace into lhe cavity is greater than lhe energy derived from formation ol water-SiOH bonds in the cavity. Typical surface microspheres may have diameters on the wder of 0.1 pm or 100 nm, H--O bond distance is 0.1 nm and me Si-0 bond distance is 0.16 while nm.
514
Journal of Chemical Education
water does wet the surface of Magic Sand. Water apparently can be forced to enter the cavities of Magic Sand to find a hospitable, polar environment there, because when vacuum is once more applied, the sand stays wetted for the most part. Surprisingly, however, commercially available bonded phase material (Supelco LC-18) behaves about the same way, presumably because i t too has unreacted silanol groups. Water minimizes the area of the interface that is created in the presence of Magic Sand particles by forcing them t o coalesce, ideally into spheres, but also into columns and other shapes when forces other than surface tension are onerative. The formation of nonwetted shapes in Maeic Sand appears similar to droplet formation in suspensions of hvdrocarhons and water. Formation of visible droplets of a hGdrophobic oil does not appear to be associated-with the large entropy change that is usually considered responsible for the classic hydrophobic effect (8). It is probably wise to emphasize that Magic Sand is a model of the hydrophohic effect, in the sensethat grains of the sand model the way hydrophobic molecules tend to associate in water. The Magic Sand model fails for interesting amphiphilic molecules like phospholipids, which have both hydrophobic and hydronhilic moities. because the interaction between Maeic S a n i and water does not lead to the specific forms associatled with ampbophiles, like micelles and bilayers. The shapes assumed by Magic Sand are quite variable. Nonetheless, the forms that Magic Sand assumes in water suggest that hydrophobic interactions are critical to biogenesis, as Harold Morowitz says in "Mayonnaise and the Origin of Life" (9). The formation of vesicles with bilayer membranes constructed from amphipbiles is critical to the origins of life; formation of vegetable oil spherules coated with egg-yolk phospholipids is critical to the production of good mayonnaise. Hoth depend upon insolubility in an aqueous environment.
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The Solvophoblc Effect It is interesting to investigate the true hydrophobic effect in liauidsother than water. whereit is called the solvonhobic effect. Several authors have suggested that bulk surfLce tension of solvents correlates well with the solvo~hobiceffect (10) and may in fact explain it. I t is clear f r o r n ~ i ~ u1 r ethat aeglomeration on the molecular scale must decrease surface &aof the surrounding liquid as well as increase its entropy. The large curvature of the molecular scale cavity . may . or may not be &pifieant. When Magic Sand is added to liquids other than water, it exhibits bulk nonwettability in liquids with high surface tensions (see table) including formamide, glycerine, and ethylene glycol. A solution of more than 25%methanol in water (with a surface tension of less than about 46 dyneslcm) does not exhibit the effect, nor does a solution of more than 55% dioxanein water (with asurface tension of less than about 40 dyneslcm). Water, near its boding polnt, has a ~urfacetenswn about the same as formamide. and M a e ~ cSand is slowlv wet by water above 90 OC. ~ m m o n i asolutibns produce pe;piexing results. The effect aonears to depend on the order of addition of concentrated a&eous ammonia and water to Magic Sand. The behavior of Magic Sand appears to parallel the bebavior of truly hydrophobic substances, and the parallel mav suggest that the surface-tension driving force is a t least important complement to the entropy driving force, which has traditionally been used to explain the hydrophobic effect. Reversed-Phase HPLC Traditional or "normal-phase" liquid chromatography depends on the affinity of polar eluites for a polar (typically silica or alumina) stationary phase. Solvents of increasing polarity, ranging from hydrocarbons to water, have increas-
Eluting Strength lor a Silica Liquid Chromatography Column, Surtace Tension, and Behavior of Magic Sand In Some Common Solvent Slrength Parameter. Silica Solvent
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large
Water
Glycerol Arnmia Formamlde EthyleneGiycol Methanol Aniline Dioxane Acetophenone 'ln the last wlumn.
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1.11 0.95 0.62 0.56
0.51 "Y" indicates mat Magic Sand i3
Surface Tendon dyneslcm
Magic
72 63 62
Y Y
58 48
Y
23
N
44
N N
33 39
7
Y
N
nonwemble, as it Is In water.
~ o t elower : satuentstrsngm(s;tioacolumn) caelates wllh higher solvent hydmphobidhl. l e s tendency lor hydrophobic eliect.
ing eluting strength. In reversed-phase liquid chromatography (RPLC), the stationary phase is very nonpolar, and nonpolar liquids are the strongest eluants. The interaction between an eluite and the nonpolar stationary phase in RPLC may be largely the hydrophobic bond (11,12). The term "hvdrophobic interaction chromatography" (HIC) has describe reversed-phase e h r ~ m a t o ~ r a p hiny been usid which no additional specific interaction (such as acidbase, or charge attractions) between the eluite and stationary phase has been intentionally exploited. In RPLC, the retention time can he varied verv ureciselv bv adding an oraanic modifier t o an aqueous eluentto reduce the magnitude ifthe hvdronhohic effect. Water is the weakest eluant, since i t &om&es the aggregation of the typically lipophilic eluites with the stationary phase, but methanol or acetonitrile can reduce the hydrophobic effect dramatically, and thus increase the eluant strength. Once more, as the Magic Sand model suggests, the association would not exist in the absence of the eluant. The table gives the solvent strength parameter (13) for silica columns. The solvent strength for reversed-phase columns, or the ability of'a solvent M weaken the to ~-~~hvdroohohic effect. would be inverselv . nrouortional . the values given here. It is interesting to note the strong correlation between solvent strength and the effect with Magic Sand. Magic Sand is wettable by liquids that are strone solvents for RPLC. ~ h ; !stationary phase used in RPLC is usually synthesized hv chemicallv bonding an alkylsil~lgroup to a silica substrate by boiling the sicca in toluene 4 t h i n organochlorosilane (14). The term "bonded-phase liquid chromatography" (BPLC) is sometimes used. The silanol (Si-OH) bond is replaced with the siloxane group, as shown in Figure 4. The microspheres on the surface of Magic Sand probably have a small percentage of silanol groups derivatized in this way. The Maeic Sand demonstration if5modelstbe hvdro~hobic interaction of the eluite with'ihe nonpolar stationary nhase and shows how the interaction denends on the presence of a solvent like water. Once more i t should he emphasized that the Maeic Sand effect may arise from a different kind of interaction than the true hydrophobic effect. Magic Sand is a pellicular (coated) material consisting of silica granules coated with silica microspheres that are lightly derivatized with alkylsilanes. I t behaves as a very weak normal stationary phase, like silica or alumina, when used for liquid chromatography under slightly higher than atmospheric pressure. Magic Sand is certainly not an optimum stationary phase because of its mixed hydrophobichydronhilic ~-~ nature and relativelv small surface area with consequent low capacity. I t does weakly separate methylene blue and methvl red as a silica gel or alumina column would5. Magic add is nonwettable in bulk, like a nonpolar or hydrophobic stationary phase, hut behaves as a normal phase (polar) substance on the molecular scale in liquid chroma~~
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Figure 4. Schematic representation of the preparation of a typical reversed stationary phase by bonding a hydrophobic substance to the surface of silica. In mis case, dirnethyioctadscylchlorosII~neis used to replace most of the Surface silanol groups on a silica gel. A trichiorwrganosilane is frequently used to obtain higher surface coverage in shorter reaction times. tography. This is an important distinction. The hydrophobic groups that are bonded to RPLC stationary phases create some pores or cavities where only a very few silanol groups are accessible, but the size of the cavity is on the scale of the size of molecules, and i t is preponderantly nonpolar and hydrophobic. Cavities on RPLC stationary phases are thus different in nature from the large cavities of Magic Sand, which contain manv silanol erouos. " . . with hulk surface tension precluding the otherwise favorable interaction of water with all SiOH erouos in the cavitv. The resultine bulk nonwettability of l ~ a i i cSand is n i t accompaniei by strong microscopic hydrophobicity. These demonstrations may he useful in instrumental analvsis courses where thev can help develop a "feel" for the $;re of HP1.C s t a t i o n a j phases.For example, they show how nonwettahility of hydrophobic stationary phases causes "channelling" in col&ns. They help visualize how mass transfer can be impeded by the phase boundary caused by hydrophobic stationary phases in an aqueous eluant, leading t o broad peaks in the absence of organic modifiers. The hydrophobic effecr is also important to an understanding of micellar eluants and separations based on the secondary equilibria that are established in them (15). Note: An earlier demonstration (16) involving Magic Sand was discovered durine review of'this work. It is stated in the earlier work that ~ a i i Sand c has a coating of "colored nonoolar material" that ran be removed with organic solvents. while the color was removed by organic solvents, none was found that would reduce the hulk unwettability of Magic Sand. Probably only a strong base solution will hydrolyze bonds responsible for the effect. the Si-0-C Acknowledgment The author is grateful to the Clifford W. Estes Company for samples of Magic Sand and to Richard Hermanof Lehigh university for consultation on scanning electron micrographs. Lltereture Cited I. Robbins, T. Euen Cougiria Gal rhoB1~~~: Houghton Mifflin: Boston. 1976: p 2. 2. Tanford. C. The Hydrophobic Effect: Formation of Mieelles and fliologieol Membranes:Wiley: New York, 1973;p 1. 3. Aert8.T.; Clauarsert. J.J. Chem.Educ.1986.63, 993-995.
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5 . Ref2, pp
16-19.29.
6. Ben-Naim, A. Hydrophobic Iniemctions:Plenum: New York. 1980. 7. Tully. Paul R. etsl.,U.S.Patent#3,562.153(February9,19711. 8. Kauzmann, W . In Aduoncsr in Profsin Chemktly. Anflnson. C. B., Jr.; Anson, M. L.: Bailey, K.: Edasll, J. T., Eds.; Academic: New York. 1959:p 43. 9. Morowitz, H. J. Moyonnoisa and the O~iginof Life: Thoughts of Minds and Molecules; Seribner: New York, 1985. 10. sinanoglu,o.~n ~ o i ~ e ~ i a r ~ a s ~ c iinfliology;Pullmsn.Biiiird,Ed.;A~d~dddmii: itiini New York, 1967:p 427. 11. Kargor,B. L.;Gant,J.R.:Har*opf,A.: Weiner,P, H. J.Chromofog 1974 128.65-78. 12. Horvath, C.: Melander. W.: Molnsr, L J Chromalog. 1976.125.129-156. 13. snyder. L. R.: Kirkland, J. J. Introduction t o Modern Liputd Chromatography, 2nd ed.: Wiby: New York, 1979;p 246. 14. Major~,R.E.:Hopper,M.J. J. Chromotog.Sci.1974.12.767. Is. Uorsey, J. G. Chromofogmphy 1987, May. 13. 16. Hoffman. A. B.J Cham. Educ. 1982,39.155.
'Work demonstrating the chromatographic behavior of Magic Sand was done oy Kutztown Univers~tysldent Donald Henderson. Volume 67 Number 6 June 1990
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